Dna molecules for expression of bile salt-stimulated lipase

ABSTRACT

The invention relates to DNA molecules, recombinant vectors and cell cultures for use in methods for expression of bile salt-stimulated lipase (BSSL) in the methylotrophic yeast Pichia pastoris.

TECHNICAL FIELD

[0001] The invention relates to DNA molecules, recombinant vectors andcell cultures for use in methods for expression of bile salt-stimulatedlipase (BSSL) in the methylotrophic yeast Pichia pastoris.

BACKGROUND ART

[0002] Bile salt-stimulated lipase (BSSL; EC 3.1.1.1) (for a review seeWang & Hartsuck, 1993) accounts for the majority of the lipolyticactivity of the human milk. A characteristic feature of this lipase isthat it requires primary bile salts for activity against emulsified longchain triacylglycerols. BSSL has so far been found only in milk fromman, gorilla, cat and dog (Hernell et al., 1989).

[0003] BSSL has been attributed a critical role for the digestion ofmilk lipids in the intestine of the breastfed infant (Fredrikzon et al.,1978). BSSL is synthesized in humans in the lactating mammary gland andsecretes with milk (Bläckberg et al., 1987). It accounts forapproximately 1% of the total milk protein (Bläckberg & Hernell, 1981).

[0004] It has been suggested that BSSL is the major rate limiting factorin fat absorption and subsequent growth by, in particular premature,infants who are deficient in their own production of BSSL, and thatsupplementation of formulas with the purified enzyme significantlyimproves digestion and growth of these infants (U.S. Pat. No. 4,944,944;Oklahoma Medical Research Foundation). This is clinically important inthe preparation of infant formulas which contain relative highpercentage of triglycerides and which are based on plant or non humanmilk protein sources, since infants fed with these formulas are unableto digest the fat in the absence of added BSSL.

[0005] The cDNA structures for both milk BSSL and pancreas carboxylicester hydrolase (CEH) have been characterized (Baba et al., 1991; Huiand Kissel, 1991; Nilsson et al., 1991; Reue et al., 1991) and theconclusion has been drawn that the milk enzyme and the pancreas enzymeare products of the same gene, the CEL gene. The cDNA sequence (SEQ IDNO: 1) of the CEL gene is disclosed in U.S. Pat. No. 5,200,183 (OklahomaMedical Research Foundation); WO 91/18293 (Aktiebolaget Astra); Nilssonet al., (1990); and Baba et al., (1991). The deduced amino acid sequenceof the BSSL protein, including a signal sequence of 23 amino acids, isshown as SEQ ID NO: 2 in the Sequence Listing, while the sequence of thenative protein of 722 amino acids is shown as SEQ ID NO: 3.

[0006] The C-terminal region of the protein contains 16 repeats of 11amino acid residues each, followed by an 11 amino acid conservedstretch. The native protein is highly glycosylated and a large range ofobserved molecular weights have been reported. This can probably beexplained by varying extent of glycosylation (Abouakil et al., 1988).The N-tenninal half of the protein is homologous to acetyl cholineesterase and some other esterases (Nilsson et al., 1990).

[0007] Recombinant BSSL can be produced by expression in a suitable hostsuch as E. coli, Saccharomyces cerevisiae, or mammalian cell lines. Forthe scaling-up of a BSSL expression system to make the production costcommercially viable, utilization of heterologous expression systemscould be envisaged. As mentioned above, human BSSL has 16 repeats of 11amino acids at the C-terminal end. To determine the biologicalsignificance of this repeat region, various mutants of human BSSL havebeen constructed which lack part or whole of the repeat regions (Hanssonet al., 1993). The variant BSSL-C (SEQ ID NO: 4), for example, hasdeletions from amino acid residues 536 to 568 and from amino acidresidues 591 to 711. Expression studies, using mammalian cell line C127host and bovine papilloma virus expression vector, showed that thevarious variants can be expressed in active forms (Hansson et al.,1993). From the expression studies it was also conduded that the prolinerich repeats in human BSSL are not essential for catalytic activity orbile salt activation of BSSL. However, production of BSSL or its mutantsin a mammalian expression system could be too expensive for routinetherapeutic use.

[0008] A eukaryotic system such as yeast may provide significantadvantages, compared to the use of prokaryotic systems, for theproduction of certain polypeptides encoded by recombinant DNA. Forexample, yeast can generally be grown to higher cell densities thanbacteria and may prove capable of glycosylating expressed polypeptides,where such glycosylation is important for the biological activity.However, use of the yeast Saccharomyces cerevisiae as a host organismoften leads to poor expression levels and poor secretion of therecombinant protein (Cregg et al., 1987). The maximum levels ofheterologous proteins in S. cerevisae are in the region of 5% of totalcell protein (Kingsman et al., 1985). A further drawback of usingSacharomyces cerevisiae as a host is that the recombinant proteins tendto be overglycosylated which could affect activity of glycosylatedmammalian proteins.

[0009]Pichia pastoris is a methylotrophic yeast which can grow onmethanol as a sole carbon and energy source as it contains a highlyregulated methanol utilization pathway (Ellis et al., 1985). P. pastorisis also amenable to efficient high cell density fermentation technology.Therefore recombinant DNA technology and efficient methods of yeasttransformation have made it possible to develop P. pastoris as a hostfor expression of heterologous protein in large quantity, with amethanol oxidase promoter based expression system (Cregg et al., 1987).

[0010] Use of Pichia pastoris is known in the art as a host for theexpression of e.g. the following heterologous proteins: human tumornecrosis factor (EP-A-0263311); Bordetella pertactin antigens (WO91/15571); hepatitis B surface antigen (Cregg et al., 1987); humanlysozyme protein (WO 92/04441); aprotinin (WO 92/01048). However,successful expression of a heterologous protein in active, soluble andsecreted form depends on a variety of factors, e.g. correct choice ofsignal peptide, proper construction of the fusion junction between thesignal peptide and the mature protein, growth conditions, etc.

PURPOSE OF THE INVENTION

[0011] The purpose of the invention is to overcome the above mentioneddrawbacks with the previous systems and to provide a method for theproduction of human BSSL with is cost-effective and has a yieldcomparable with, or superior to, production in other organisms. Thispurpose has been achieved by providing methods for expression of BSSL inPichia pastoris cells.

[0012] By the invention it has thus been shown that human BSSL and thevariant BSSL can be expressed in active form secreted from P. pastoris.The native signal peptide, as well as the heterologous signal peptidederived from S. cerevisiae invertase protein, have been used totransiocate the mature protein into the culture medium as an active,properly processed form.

DESCRIPTION OF THE INVENTION

[0013] In a first aspect, the invention provides a DNA moleculecomprising:

[0014] (a) a region coding for a polypeptide which is human BSSL or abiologically active variant thereof;

[0015] (b) joined to the 5′-end of said polypeptide coding region, aregion coding for a signal peptide capable of directing secretion ofsaid polypeptide from Pichia pastoris cells transformed with said DNAmolecule; and

[0016] (c) operably-linked to said coding regions defined in (a) and(b), the methanol oxidase promoter of Pichia pastoris or a functionallyequivalent promoter.

[0017] The term “biologically active variant” of BSSL is to beunderstood as a polypeptide having BSSL activity and comprising part ofthe amino acid sequence shown as SEQ ID NO: 3 in the Sequence Listing.The term “polypeptide having BSSL activity” is in this context to beunderstood as a polypeptide comprising the following properties: (a)being suitable for oral administration; (b) being activated by specificbile-salts; and (c) acting as a non-specific lipase in the contents ofthe small intestines, i.e. being able to hydrolyze lipids relativelyindependent of their chemical structure and physical state (emulsified,micellar, soluble).

[0018] The said BSSL variant can e.g. be a variant which comprises lessthan 16 repeat units, whereby a “repeat unit” will be understood as arepeated unit of 11 amino acids, encoded by a nudeotide sequenceindicated as a “repeat unit” under the heading “(ix) FEATURE” in“INFORMATION FOR SEQ ID NO: 1” in the Sequence Listing. In particular,the BSSL variant can be the variant BSSL-C, wherein amino acids 536 to568 and 591 to 711 have been deleted (SEQ ID NO: 4 in the SequenceListing). Consequently, the DNA molecule according to the invention ispreferably a DNA molecule which encodes BSSL (SEQ ID NO: 3) or BSSL-C(SEQ ID NO: 4).

[0019] However, the DNA molecules according to the invention are not tobe limited strictly to DNA molecules which encode polypeptides withamino acid sequences identical to SEQ ID NO: 3 or 4 in the SequenceListing. Rather the invention encompasses DNA molecules which code forpolypeptides carrying modifications like substitutions, small deletions,insertions or inversions, which polypeptides nevertheless havesubstantially the biological activities of BSSL. Included in theinvention are consequently DNA molecules coding for BSSL variants asstated above and also DNA molecules coding for polypeptides, the aminoacid sequence of which is at least 90% homologous, preferably at least95% homologous, with the amino acid sequence shown as SEQ ID NO: 3 or 4in the Sequence Listing.

[0020] The signal peptide referred to above can be a peptide which isidentical to, or substantially similar to, the peptide with the aminoacid sequence shown as amino acids −20 to −1 of SEQ ID NO: 2 in theSequence Listing. Alternatively, it can be a peptide which comprises aSaccharomyces cerevisiae invertase signal peptide.

[0021] In a further aspect, the invention provides a vector comprising aDNA molecule as defined above. Preferably, such a vector is a replicableexpression vector which carries and is capable of mediating expression,in a cell of the genus Pichia, of a DNA sequence coding for human BSSLor a biologically active variant thereof. Such a vector can e.g. be theplasmid vector pARC 5771 (NCIMB 40721), pARC 5799 (NCIMB 40723) or pARC5797 (NCIMB 40722).

[0022] In another aspect, the invention provides a host cell culturecomprising cells of the genus Pichia transformed with a DNA molecule ora vector as defined above. Preferably, the host cells are Pichiapastoris cells of a strain such as PPF-1 or GS115. The said cell culturecan e.g. be the culture PPF-1[pARC 5771] (NCIMB 40721), GS115[pARC 5799](NCIMB 40723) or GS115[pARC 5797] (NCIMB 40722).

[0023] In yet another aspect, the invention provides a process theproduction of a polypeptide which is human BSSL, or a biologicallyactive variant thereof, which comprises culturing host cells accordingto the invention under conditions whereby said polypeptide is secretedinto the culture medium, and recovering said polypeptide from theculture medium.

EXAMPLES OF THE INVENTION Example 1 Expression of BSSL in Pichiapastoris PPF-1

[0024] 1.1. Construction of pARC 0770

[0025] The cDNA sequence (SEQ ID NO: 1) coding for the BSSL protein,including the native signal peptide (below referred to as NSP) wascloned in pTZ19R (Pharmacia) as an EcoRI-SacI fragment. The cloning ofNSP-BSSL cDNA into S. cerevisiae expression vector pSCW 231 (obtainedfrom professor L. Prakash, University of Rochester, N.Y., USA), which isa low copy number yeast expression vector wherein expression is undercontrol of the constitutive ADH1 promoter, was achieved in two steps.Initially the NSP-BSSL cDNA was cloned into pYES 2.0 (Invitrogen, USA)as an EcoRI-SphI fragment from pTZ19R-SP-BSSL. The excess 89 base pairsbetween the EcoRI and NcoI at the beginning of the signal peptide codingsequence were removed by creating an EcoRI/NcoI (89) fusion andregenerating an EcoRI site. The resulting clone pARC 0770 contained anATG codon, originally encoded within the NcoI site which was immediatelyfollowed by the regenerated EcoRI site in frame with the remainingNSP-BSSL sequence.

[0026] 1.2. Construction of pARC 5771 Plasmid

[0027] To construct a suitable expression vector for the expression ofBSSL, the cDNA fragment encoding the BSSL protein along with its nativesignal peptide was cloned with P. pastoris expression vector pDM 148.The vector pDM 148 (received from Dr. S. Subramani, UCSD) wasconstructed as follows: the upstream untranslated region (5′-UTR) andthe down stream untranslated region (3′-UTR) of methanol oxidase (MOX1)gene were isolated by PCR and placed in tandem in the multiple cloningsequence (MCS) of E. coli vector pSK⁺ (available from Stratagene, USA).

[0028] For proper selection of the putative P. pastoris transformants, aDNA sequence coding for S. cerevisiae ARG4 gene along with its ownpromoter sequence was inserted between the 5′- and the 3′-UTR in pSK−.The resulting construct pDM148 has following features: in the MCS regionof pSK− the 5′-UTR of MOX, S. cerevisiae ARG4 genomic sequence and the3′-UTR of MOX were cloned. Between the 5′-UTR of MOX and the ARG4genomic sequence a series of unique restriction sites (SalI, ClaI,EcoRI, PstI, SmaI and BamHI) were situated where any heterologousprotein coding sequence can be cloned for expression under the controlof the MOX promoter in P. pastoris. To facilitate integration of thisexpression cassette into the MOX1 locus in P. pastoris chromosome, theexpression cassette can be cleaved from the rest of the pSK⁻ vector bydigestion with NotI restriction enzyme.

[0029] The 5′-UTR of MOX1 of P. pastoris cloned in pDM 148 was about 500bp in length while the 3′-UTR of MOX1 from P. pastoris cloned into pDM148 was about 1000 bp long. To insert the NSP-BSSL cDNA sequence,between the 5′-UTR of MOX1 and the S. cerevisiae ARG4 coding sequence inpDM 148, the cDNA insert (SP-BSSL) was isolated from pARC 0770 bydigestion with EcoRI and BamHI (approximately 2.2 kb DNA fragment) andcloned between the EcoRI and BamHI sites in pDM 148.

[0030] The resulting construct pARC 5771 (NCIMB 40721) contained the P.pastoris MOX1 5′-UTR followed by the NSP-BSSL coding sequence followedby S. cerevisiae ARG4 gene sequence and 3′-UTR of MOX1 gene of P.pastoris while the entire DNA segment from 5′-UTR of MOX1 to the 3′-UTRof MOX1 was cloned at the MCS of pSK−.

[0031] 1.3. Transformation of BSSL in P. pastoris Host PPF-1

[0032] For expression of BSSL in P. pastoris host PPF-1 (his4, arg4;received from Phillips Petroleum Co.), the plasmid pARC 5771 wasdigested with NotI and the entire digested mix (10 μg of total DNA) wasused to transform PPF-1. The transformation protocol followed wasessentially the yeast spheroplast method described by Cregg et al.(1987). Transformants were regenerated on minimal medium lackingarginine so that Arg+ colonies could be selected. The regeneration topagar containing the transformants was lifted and homogenized in waterand yeast cells plated to about 250 colonies per plate on minimalglucose plates lacking arginine. Mutant colonies are then identified byreplica plating onto minimal methanol plates. Approximately 15% of alltransformants turned out to be Mut^(s) (methanol slow growing)phenotype.

[0033] 1.4. Screening for Transformants Expressing BSSL

[0034] In order to screen large number of transformants rapidly for theexpression of lipase a lipase plate assay method was developed. Theprocedure for preparing these plates was as follows: to a solution of 2%agarose (final), 10×Na-cholate solution in water was added to a finalconcentration of 1%. The lipid substrate trybutine was added in themixture to a final concentration of 1% (v/v). To support growth of thetransformants the mixture was further supplemented with 0.25% yeastnitrogen base (final) and 0.5% methanol (final). The ingredients weremixed properly and poured into plates up to 3-5 mm thickness. Once themixture became solid, the transformants were streaked onto the platesand the plates were further incubated at +37° C. for 12 h. The lipaseproducing clones showed a clear halo around the clone. In a typicalexperiment 7 out of a total of 93 transformants were identified as BSSLproducing transformants. Two clones (Nos. 39 and 86) producing thelargest halos around the streaked colony were picked out for furthercharacterization.

[0035] 1.5. Expression of BSSL from PPF-1[pARC 5771]

[0036] The two transformants Nos. 39 and 86 described in Section 1.4were picked out and grown in BMGY liquid media (1% yeast extract, 2%bactopeptone, 1.34% yeast nitrogen base without amino acid, 100 mM KPO₄buffer, pH 6.0, 400 μg/l biotin, and 2% glycerol) for 24 h at 30° C.until the cultures reached A₆₀₀ close to 40. The cultures were pelleteddown and resuspended in BMMY (2% glycerol replaced by 0.5% methanol inBMGY) media at A₆₀₀=300. The induced cultures were incubated at 30° C.with shaking for 120 h. The culture supernatants were withdrawn atdifferent time points for the analysis of the expression of BSSL byenzyme activity assay, SDS-PAGE analysis and western blotting.

[0037] 1.6. Detection of BSSL Enzyme Activity in the CultureSupernatants of Clone Nos. 39 and 86

[0038] To determine the enzyme activity in the cell free culturesupernatant of the induced cultures Nos. 39 and 86 as described inSection 1.5, the cultures were spun down and 2 μl of the cell freesupernatant was assayed for BSSL enzyme activity according to the methoddescribed by Hernell and Olivecrona (1974). As shown in Table 1, boththe cultures were found to contain BSSL enzyme activity with the maximumactivity at 96 h following induction.

[0039] 1.7. Western Blot Analysis of Culture Supernatants of PPF-1:pARC5771 Transformants (Nos. 39 and 86)

[0040] To determine the presence of recombinant BSSL in the culturesupernatants Nos. 39 and 86 of PPF-1[pARC 5771] transformants, thecultures were grown and induced as described in Section 1.5. Thecultures were withdrawn at different time points following induction andsubjected to Western blot analysis using anti BSSL polyclonal antibody.The results indicated the presence of BSSL in the culture supernatant asa 116 kDa band.

Example 2 Expression of BSSL in Pichia pastoris GS115

[0041] 2.1. Construction of pARC 5799

[0042] Since the 5′-MOX UTR and 3′-MOX UTR were not properly defined andsince the pDM 148 vector lacks any other suitable marker (e.g. a G418resistance gene) to monitor the number of copies of the BSSL integratedin the Pichia chromosome, the cDNA insert of native BSSL along with itssignal peptide was cloned into another P. pastoris expression vector,pHIL D4. The integrative plasmid pHIL D4 was obtained from PhillipsPetroleum Company. The plasmid contained 5′-MOX1, approximately 1000 bpsegment of the alcohol oxidase promoter and a unique EcoRI doning site.It also contained approximately 250 bp of 3′-MOX1 region containingalcohol oxidase terminating sequence, following the EcoRI site. The“termination” region was followed by P. pastoris histidinoldehydrogenase gene HIS4 contained on a 2.8 kb fragment to complement thedefective HIS4 gene in the host GS115 (see below). A 650 bp regioncontaining 3′-MOX1 DNA was fused at the 3′-end of HIS4 gene, whichtogether with the 5′-MOX1 region was necessary for site-directedintegration. A bacterial kanamycin resistance gene from pUC4K(PL-Biochemicals) was inserted at the unique NaeI site between HIS4 and3′-MOX1 region at 3′ of the HIS4 gene.

[0043] To clone the NSP-BSSL coding cDNA fragment at the unique EcoRIsite of pHIL D4, a double stranded oligo linker having a BamHI-EcoRIcleaved position was ligated to the BamHI digested plasmid pARC 5771 andthe entire NSP-BSSL coding sequence was pulled out as a 2.2 kb EcoRIfragment. This fragment was cloned at the EcoRI site of pHIL D-4 and thecorrectly oriented plasmid was designated as pARC 5799 (NCIMB 40723).

[0044] 2.2. Transformation of pARC 5799

[0045] To facilitate integration of the NSP-BSSL coding sequence at thegenomic locus of MOX1 in P. pastoris the plasmid pARC 5799 was digestedwith BglII and used for transformation of P. pastoris strain GS115(his4)(Phillips Petroleum Company) according to a protocol described inSection 1.5. In this case, however, the selection was for Hisprototrophy. The transformants were picked up following serial dilutionplating of the regenerated top agar and tested directly for lipase plateassay as described in Section 1.4. Two transformant clones (Nos. 9 and21) were picked up on the basis of the halo size on the lipase assayplate and checked further for the expression of BSSL. The clones werefound to be Mut⁺.

[0046] 2.3. Determination of BSSL Enzyme Activity in the CultureSupernatants of GS115[pARC 5799] Transformants Nos. 9 and 21.

[0047] The two transformed clones Nos. 9 and 21 of GS115[pARC 5799] weregrown essentially following the protocol described in Section 1.5. Theculture supernatants at different time points following induction wereassayed for BSSL enzyme activity as described in Section 1.6. As shownin Table 1, both the culture supernatants were found to contain BSSLenzyme activity and the enzyme activity was highest after 72 h ofinduction. Both clones showed a superior expression of BSSL compared tothe clones of PPF-1[pARC 5771].

[0048]2.4. SDS-PAGE and Western Blot Analysis of Culture Supernatants ofGS115[pARC 5799] Transformants Nos. 9 and 21

[0049] The culture supernatants collected at different time points, asdescribed in Section 2.3 were subjected to SDS-PAGE and western blotanalysis. From the SDS-PAGE profile it was estimated that about 60-75%of the total protein present in the culture supernatants of the inducedcultures was BSSL. The molecular weight of the protein was about 116kDa. The western blot data also confirmed that the major protein presentin the culture supernatant was BSSL. The protein apparently had the samemolecular weight as the native BSSL.

Example 3 Scaling-Up of BSSL Expression

[0050] 3.1. Scaling-up of Expression of BSSL from the Transformed CloneGS115[pARC 5799] (No. 21)

[0051] A 23 l capacity B. Braun fermenter was used. Five liters ofmedium containing, 1% YE, 2% Peptone, 1.34 YNB and 4% w/v glycerol wasautoclaved at 121° C. for 30 mm and biotin (400 μg/L finalconcentration) was added during inoculation after filter sterilization.For inoculum, glycerol stock of GS115[pARC 5799] (No. 21) inoculatedinto a synthetic medium containing YNB (67%) plus 2% glycerol (150 ml)and grown at +30° C. for 36 h was used. Fermentation conditions were asfollows: the temperature was +30° C.; pH 5.0 was maintained using 3.5 NNH₄OH and 2 N HCl; dissolved oxygen from 20 to 40% of air saturation;polypropylene glycol 2000 was used as antifoam.

[0052] Growth was monitored at regular intervals by taking OD at 600 nm.A₆₀₀ reached a maximum of 50-60 in 24 h. At this point, the batch growthphase was over as indicated by the increased dissolved oxygen levels.

[0053] Growth phase was immediately followed by the induction phase.During this phase, methanol containing 12 ml/L PTM1 salts was fed.Methanol feed rate was 6 μl/h during first 10-12 h after which it wasincreased gradually in 6 ml/h increments every 7-8 h to a maximum of 36ml/h. Ammonia used for pH control acted as a nitrogen source. Methanolaccumulation was checked every 6-8 h by using dissolved oxygen spikingand it was found to be limiting during the entire phase of induction. ODat 600 nm increased from 50-60 to 150-170 during 86 h of methanol feed.Yeast extract and peptone were added every 24 h to make final conc. of0.25% and 0.5% respectively.

[0054] Samples were withdrawn at 24 h interval and checked for BSSLenzyme activity in the cell free broth. The broth was also subjected toSDS-PAGE and western blotting analysis.

[0055] 3.2. Protein Analysis of the Secreted BSSL from the FermenterGrown Culture GS115[pARC 5799] (No. 21)

[0056] BSSL enzyme activity in cell free broth increased from 40-70 mg/l(equivalent of native protein) in 24 h to a maximum 200-227.0 mg/l(equivalent of native protein) at the end of 86-90 h. SDS-PAGE analysisof the cell free broth shows a prominent coomassie blue stained band ofmol.wt. of 116 kDa. The identity of the band was confirmed by Westernblot performed as described in Section 1.7 for native BSSL.

[0057] 3.3. Purification of Recombinant BSSL Secreted into the CultureSupernatant of GS115[pARC 5799] (No. 21) Clones

[0058] The P. pastoris clone GS115[pARC 5799] was grown and induced inthe fermenter as described in Section 3.1. For purification ofrecombinant BSSL, 250 ml of culture medium (induced for 90 h) was spunat 12,000× g for 30 minutes to remove all particulate matter. The cellfree culture supernatant was ultra filtered in an Amicon set up using a10 kDa cut off membrane. Salts and low molecular weight proteins andpeptides of the culture supernatant were removed by repeated dilutionduring filtration. The buffer used for such dilution was 5 mM BarbitolpH 7.4. Following concentration of the culture supernatant, theretentate was reconstituted to 250 ml using 5 mM Barbitol, pH 7.4 and 50mM NaCl and loaded onto a Heparin-Sepharose column (15 ml bed volume)which was pre-equilibrated with the same buffer. The sample loading wasdone at a flow rate of 10 ml/hr. Following loading the column was washedwith 5 mM Barbitol, pH 7.4 and 0.1 M NaCl (200 μl washing buffer) tillthe absorbance at 250 nm reached below detection level. The BSSL waseluted with 200 ml of Barbitol buffer (5 mM, pH 7.4) and a lineargradient of NaCl ranging from 0.1 M to 0.7 M. Fractions (2.5 ml) werecollected and checked for the eluted protein by monitoring theabsorbance at 260 nm. Fractions containing protein were assayed for BSSLenzyme activity. Appropriate fractions were analyzed on 8.0% SDS-PAGE tocheck thee purification profile.

[0059] 3.4. Characterization of Purified Recombinant BSSL Secreted inthe Culture Supernatant of GS115[pARC 5799]

[0060] SDS-PAGE and Western blot analysis of the fractions (described inSection 3.3) showing maximal BSSL enzyme activity demonstrated that therecombinant protein was approximately 90% pure. The molecular weight ofthe purified protein was about 116 kDa as determined by SDS-PAGE andwestern blot analysis. When the samples were overloaded for SDS-PAGEanalysis a low molecular weight protein band could be detected byCoomassie Brilliant Blue staining which was not picked up on Westernblot. The purified protein was subjected to N-terminal analysis in anautomated protein sequencer. The results showed that the protein wasproperly processed from the native signal peptide and the recombinantprotein has the N-terminal sequence A K L G A V Y. The specific activityof the purified recombinant protein was found to be similar to that ofthe native protein.

Example 4 Expression of BSSL-C in Pichia pastoris GS115

[0061] 4.1. Construction of pARC 5797

[0062] The cDNA coding sequence for the BSSL variant BSSL-C was fused atits 5′-end with the signal peptide coding sequence of S. cerevisiae SUC2gene product (invertase), maintaining the integrity of the open readingframe initiated at the first ATG codon of invertase signal peptide. Thisfusion gene construct was initially cloned into the S. cerevisiaeexpression vector pSCW 231 (pSCW 231 is a low copy number yeastexpression vector and the expression is under the control of theconstitutive ADH1 promoter) between EcoRI and BamHI site to generate theexpression vector pARC 0788.

[0063] The cDNA of the fusion gene was further subdoned into P. pastorisexpression vector pDM 148 (described in Section 1.2) by releasing theappropriate 1.8 kb fragment by EcoRI and BamHI digestion of pARC 0788and subcloning the fragment into pDM 148 digested with EcoRI and BamHI.The resulting construct pARC 5790 was digested with BamHI. and a doublestranded oligonudeotide linker of the physical structureBamHI-EcoRI-BamHI was ligated to generate the construct pARC 5796essentially to isolate the cDNA fragment of the fusion gene, followingthe strategy as described in Section 2.1.

[0064] Finally the 1.8 kb fragment containing the invertase signalpeptide/BSSL-C fusion gene was released from pARC 5796 by EcoRIdigestion and cloned into pHIL D4 at the EcoRI site. By appropriaterestriction analysis of the expression vector containing the insert inthe proper orientation was identified and was designated as pARC 5797(NCIMB 40722).

[0065] 4.2. Expression of Recombinant BSSL-C from P. pastoris

[0066] To express recombinant BSSL-C from P. pastoris, the P. pastorishost GS115 was transformed with pARC 5797 by the method as described inSections 1.3 and 2.2. Transformants were checked for lipase productionby the method described in Sections 1.4 and 2.2. A single transformant(No. 3) was picked on the basis of high lipase producing ability by thelipase plate assay detection method and was further analyzed forproduction of BSSL enzyme activity in the culture supernatant byessentially following the method as described in Sections 1.6 and 2.3.As shown in Table 1, the culture supernatant of GS115[pARC 5797] (No. 3)contained BSSL enzyme activity and the amount increased progressivelytill 72 h following induction.

[0067] 4.3. SDS-PAGE and Western Blot Analysis of Culture Supernatant ofGS115[pARC 5797] Transformant (No. 3)

[0068] The culture supernatant collected at various time points asdescribed in Section 4.2 were subjected to SDS-PAGE and western blotanalysis as described in Sections 1.7 and 2.4. From the SDS-PAGE profileit was estimated that about 75-80% of the total extracellular proteinwas BSSLC. The molecular weight of the protein as estimated fromSDS-PAGE analysis was approximately 66 kDa. On western blot analysisonly two bands (doublet) around 66 kDa were found to be immunoreactiveand thus confirming the expression of recombinant BSSL-C.

Example for Comparison Expression of BSSL in S. cerevisiae

[0069] Attempts to express BSSL in Saccharomyces cerevisiae were made.BSSL was poorly secreted in S. cerevisiae and the native signal peptidedid not work efficiently. In addition, the native signal peptide did notget cleaved from the mature protein in S. cerevisiae.

REFERENCES

[0070] Abouakil, N., Rogalska, E., Bonicel, J. and Lombardo, D. (1988)Biochim. Biophys. Acta. 961, 299-308.

[0071] Baba, T., Downs, D., Jackson, K. W., Tang, J. and Wang, C -S(1991) Biochemistry 30, 500-510.

[0072] Bläckberg, L. and Hernell, O. (1981) Eur. J. Biochem. 116,221-225.

[0073] Bläckberg, L., Ängquist, K. A. and Hernell, O. (1987) FEBS Lett.217, 37-41.

[0074] Cregg, J. M. et al. (1987) Bio/Technology 5, 479-485.

[0075] Ellis, S. B. et al. (1985) Mol. Cell. Biol. 5, 1111-1121.

[0076] Fredrikzon, B., Hernell, O., Bläckberg, L. and Olivecrona, T.(1978) Pediatric Res. 12, 1048-1052.

[0077] Hansson, L., Bläckberg, L., Edlund, M., Lundberg, L., Strömqvist,M. and Hernell, O. (1993) J. Biol. Chem. 268, 26692-26698.

[0078] Hernell, O . and Olivecrona, T. (1974) Biochim. Biophys. Acta369, 234-244.

[0079] Hernell, O., Bläckberg, L and Olivecrona, T. (1989) in: Textbookof gastroenterology and nutrition in infancy (Lebenthal, E., ed.)347-354, Raven Press, NY.

[0080] Hernell, O. and Bläckberg, L. (1982) Pediatric Res. 16, 882-885.

[0081] Hui, D. Y. and Kissel, J. A. (1990) FEBS Letters 276, 131-134.

[0082] Kingsman, et. al. (1985) Biotechnology and Genetic EngineeringReviews 3, 377-416.

[0083] Nilsson, J., Bläckberg, L., Carlsson, P., Enerbäck, S., Hernell,O. and Bjursell, G. (1990) Eur. J. Biochem. 192, 543-550.

[0084] Reue, K., Zambaux, J., Wong, H., Lee, G., Leete, T. H., Ronk, M.,Shively, J. E., Sternby, B., Borgström, B., Ameis, D. and Scholtz, M. C.(1991) J. Lipid. Res. 32, 267-276.

[0085] Wang, C-S, and Hartsuck, J. A. (1993) Biochim. Biphys Acta 1166,1-19.

[0086] Deposit Of Microorganisms

[0087] The following plasmids, transformed into Pichia pastoriscultures, have been deposited under the Budapest Treaty at the NationalCollections of Industrial and Marine Bacteria (NCIMB), Aberdeen,Scotland, UK. The date of deposit is May 2, 1995. Strain[plasmid] NCIMBNo. PPF-1[pARC 5771] 40721 GS115[pARC 5799] 40723 GS115[pARC 5797] 40722

[0088] TABLE 1 Enzyme activity in the culture supernatants of Pichiapastoris transformants. Enzyme activity in mg/L equivalent of nativeBSSL PPF- GS115 Hours after 1[pARC 5771] [pARC 5799] GS115[pARC 5797]induction No.39 No.86 No.9 No.21 No.3 24 0.254 0.135 1.53 1.72 0.37 482.69 3.12 17.28 34.70 40.9 72 3.96 8.25 37.37 50.60 44.9 96 11.26 13.6026.34 50.60 35.6 120 8.42 13.13 13.60 22.30 17.8

[0089]

1 4 2428 base pairs nucleic acid double linear cDNA to mRNA NO NO Homosapiens mammary gland CDS 82..2319 /product= “bile-salt-stimulatedlipase” exon 985..1173 exon 1174..1377 exon 1378..1575 exon 1576..2415mat_peptide 151..2316 polyA_signal 2397..2402 repeat_region 1756..22835′UTR 1..81 repeat_unit 1756..1788 repeat_unit 1789..1821 repeat_unit1822..1854 repeat_unit 1855..1887 repeat_unit 1888..1920 repeat_unit1921..1953 repeat_unit 1954..1986 repeat_unit 1987..2019 repeat_unit2020..2052 repeat_unit 2053..2085 repeat_unit 2086..2118 repeat_unit2119..2151 repeat_unit 2152..2184 repeat_unit 2185..2217 repeat_unit2218..2250 repeat_unit 2251..2283 Jeanette Blackberg, Lars Carlsson,Peter Enerback, Sven Hernell, Olle Bjursell, Gunnar Nilsson cDNA cloningof human-milk bile-salt-stimulated lipase and evidence for its identityto pancreatic carboxylic ester hydrolase Eur. J. Biochem. 192 543-550Sept.-1990 1 ACCTTCTGTA TCAGTTAAGT GTCAAGATGG AAGGAACAGC AGTCTCAAGATAATGCAAAG 60 AGTTTATTCA TCCAGAGGCT G ATG CTC ACC ATG GGG CGC CTG CAACTG GTT 111 Met Leu Thr Met Gly Arg Leu Gln Leu Val -23 -20 -15 GTG TTGGGC CTC ACC TGC TGC TGG GCA GTG GCG AGT GCC GCG AAG CTG 159 Val Leu GlyLeu Thr Cys Cys Trp Ala Val Ala Ser Ala Ala Lys Leu -10 -5 1 GGC GCC GTGTAC ACA GAA GGT GGG TTC GTG GAA GGC GTC AAT AAG AAG 207 Gly Ala Val TyrThr Glu Gly Gly Phe Val Glu Gly Val Asn Lys Lys 5 10 15 CTC GGC CTC CTGGGT GAC TCT GTG GAC ATC TTC AAG GGC ATC CCC TTC 255 Leu Gly Leu Leu GlyAsp Ser Val Asp Ile Phe Lys Gly Ile Pro Phe 20 25 30 35 GCA GCT CCC ACCAAG GCC CTG GAA AAT CCT CAG CCA CAT CCT GGC TGG 303 Ala Ala Pro Thr LysAla Leu Glu Asn Pro Gln Pro His Pro Gly Trp 40 45 50 CAA GGG ACC CTG AAGGCC AAG AAC TTC AAG AAG AGA TGC CTG CAG GCC 351 Gln Gly Thr Leu Lys AlaLys Asn Phe Lys Lys Arg Cys Leu Gln Ala 55 60 65 ACC ATC ACC CAG GAC AGCACC TAC GGG GAT GAA GAC TGC CTG TAC CTC 399 Thr Ile Thr Gln Asp Ser ThrTyr Gly Asp Glu Asp Cys Leu Tyr Leu 70 75 80 AAC ATT TGG GTG CCC CAG GGCAGG AAG CAA GTC TCC CGG GAC CTG CCC 447 Asn Ile Trp Val Pro Gln Gly ArgLys Gln Val Ser Arg Asp Leu Pro 85 90 95 GTT ATG ATC TGG ATC TAT GGA GGCGCC TTC CTC ATG GGG TCC GGC CAT 495 Val Met Ile Trp Ile Tyr Gly Gly AlaPhe Leu Met Gly Ser Gly His 100 105 110 115 GGG GCC AAC TTC CTC AAC AACTAC CTG TAT GAC GGC GAG GAG ATC GCC 543 Gly Ala Asn Phe Leu Asn Asn TyrLeu Tyr Asp Gly Glu Glu Ile Ala 120 125 130 ACA CGC GGA AAC GTC ATC GTGGTC ACC TTC AAC TAC CGT GTC GGC CCC 591 Thr Arg Gly Asn Val Ile Val ValThr Phe Asn Tyr Arg Val Gly Pro 135 140 145 CTT GGG TTC CTC AGC ACT GGGGAC GCC AAT CTG CCA GGT AAC TAT GGC 639 Leu Gly Phe Leu Ser Thr Gly AspAla Asn Leu Pro Gly Asn Tyr Gly 150 155 160 CTT CGG GAT CAG CAC ATG GCCATT GCT TGG GTG AAG AGG AAT ATC GCG 687 Leu Arg Asp Gln His Met Ala IleAla Trp Val Lys Arg Asn Ile Ala 165 170 175 GCC TTC GGG GGG GAC CCC AACAAC ATC ACG CTC TTC GGG GAG TCT GCT 735 Ala Phe Gly Gly Asp Pro Asn AsnIle Thr Leu Phe Gly Glu Ser Ala 180 185 190 195 GGA GGT GCC AGC GTC TCTCTG CAG ACC CTC TCC CCC TAC AAC AAG GGC 783 Gly Gly Ala Ser Val Ser LeuGln Thr Leu Ser Pro Tyr Asn Lys Gly 200 205 210 CTC ATC CGG CGA GCC ATCAGC CAG AGC GGC GTG GCC CTG AGT CCC TGG 831 Leu Ile Arg Arg Ala Ile SerGln Ser Gly Val Ala Leu Ser Pro Trp 215 220 225 GTC ATC CAG AAA AAC CCACTC TTC TGG GCC AAA AAG GTG GCT GAG AAG 879 Val Ile Gln Lys Asn Pro LeuPhe Trp Ala Lys Lys Val Ala Glu Lys 230 235 240 GTG GGT TGC CCT GTG GGTGAT GCC GCC AGG ATG GCC CAG TGT CTG AAG 927 Val Gly Cys Pro Val Gly AspAla Ala Arg Met Ala Gln Cys Leu Lys 245 250 255 GTT ACT GAT CCC CGA GCCCTG ACG CTG GCC TAT AAG GTG CCG CTG GCA 975 Val Thr Asp Pro Arg Ala LeuThr Leu Ala Tyr Lys Val Pro Leu Ala 260 265 270 275 GGC CTG GAG TAC CCCATG CTG CAC TAT GTG GGC TTC GTC CCT GTC ATT 1023 Gly Leu Glu Tyr Pro MetLeu His Tyr Val Gly Phe Val Pro Val Ile 280 285 290 GAT GGA GAC TTC ATCCCC GCT GAC CCG ATC AAC CTG TAC GCC AAC GCC 1071 Asp Gly Asp Phe Ile ProAla Asp Pro Ile Asn Leu Tyr Ala Asn Ala 295 300 305 GCC GAC ATC GAC TATATA GCA GGC ACC AAC AAC ATG GAC GGC CAC ATC 1119 Ala Asp Ile Asp Tyr IleAla Gly Thr Asn Asn Met Asp Gly His Ile 310 315 320 TTC GCC AGC ATC GACATG CCT GCC ATC AAC AAG GGC AAC AAG AAA GTC 1167 Phe Ala Ser Ile Asp MetPro Ala Ile Asn Lys Gly Asn Lys Lys Val 325 330 335 ACG GAG GAG GAC TTCTAC AAG CTG GTC AGT GAG TTC ACA ATC ACC AAG 1215 Thr Glu Glu Asp Phe TyrLys Leu Val Ser Glu Phe Thr Ile Thr Lys 340 345 350 355 GGG CTC AGA GGCGCC AAG ACG ACC TTT GAT GTC TAC ACC GAG TCC TGG 1263 Gly Leu Arg Gly AlaLys Thr Thr Phe Asp Val Tyr Thr Glu Ser Trp 360 365 370 GCC CAG GAC CCATCC CAG GAG AAT AAG AAG AAG ACT GTG GTG GAC TTT 1311 Ala Gln Asp Pro SerGln Glu Asn Lys Lys Lys Thr Val Val Asp Phe 375 380 385 GAG ACC GAT GTCCTC TTC CTG GTG CCC ACC GAG ATT GCC CTA GCC CAG 1359 Glu Thr Asp Val LeuPhe Leu Val Pro Thr Glu Ile Ala Leu Ala Gln 390 395 400 CAC AGA GCC AATGCC AAG AGT GCC AAG ACC TAC GCC TAC CTG TTT TCC 1407 His Arg Ala Asn AlaLys Ser Ala Lys Thr Tyr Ala Tyr Leu Phe Ser 405 410 415 CAT CCC TCT CGGATG CCC GTC TAC CCC AAA TGG GTG GGG GCC GAC CAT 1455 His Pro Ser Arg MetPro Val Tyr Pro Lys Trp Val Gly Ala Asp His 420 425 430 435 GCA GAT GACATT CAG TAC GTT TTC GGG AAG CCC TTC GCC ACC CCC ACG 1503 Ala Asp Asp IleGln Tyr Val Phe Gly Lys Pro Phe Ala Thr Pro Thr 440 445 450 GGC TAC CGGCCC CAA GAC AGG ACA GTC TCT AAG GCC ATG ATC GCC TAC 1551 Gly Tyr Arg ProGln Asp Arg Thr Val Ser Lys Ala Met Ile Ala Tyr 455 460 465 TGG ACC AACTTT GCC AAA ACA GGG GAC CCC AAC ATG GGC GAC TCG GCT 1599 Trp Thr Asn PheAla Lys Thr Gly Asp Pro Asn Met Gly Asp Ser Ala 470 475 480 GTG CCC ACACAC TGG GAA CCC TAC ACT ACG GAA AAC AGC GGC TAC CTG 1647 Val Pro Thr HisTrp Glu Pro Tyr Thr Thr Glu Asn Ser Gly Tyr Leu 485 490 495 GAG ATC ACCAAG AAG ATG GGC AGC AGC TCC ATG AAG CGG AGC CTG AGA 1695 Glu Ile Thr LysLys Met Gly Ser Ser Ser Met Lys Arg Ser Leu Arg 500 505 510 515 ACC AACTTC CTG CGC TAC TGG ACC CTC ACC TAT CTG GCG CTG CCC ACA 1743 Thr Asn PheLeu Arg Tyr Trp Thr Leu Thr Tyr Leu Ala Leu Pro Thr 520 525 530 GTG ACCGAC CAG GAG GCC ACC CCT GTG CCC CCC ACA GGG GAC TCC GAG 1791 Val Thr AspGln Glu Ala Thr Pro Val Pro Pro Thr Gly Asp Ser Glu 535 540 545 GCC ACTCCC GTG CCC CCC ACG GGT GAC TCC GAG ACC GCC CCC GTG CCG 1839 Ala Thr ProVal Pro Pro Thr Gly Asp Ser Glu Thr Ala Pro Val Pro 550 555 560 CCC ACGGGT GAC TCC GGG GCC CCC CCC GTG CCG CCC ACG GGT GAC TCC 1887 Pro Thr GlyAsp Ser Gly Ala Pro Pro Val Pro Pro Thr Gly Asp Ser 565 570 575 GGG GCCCCC CCC GTG CCG CCC ACG GGT GAC TCC GGG GCC CCC CCC GTG 1935 Gly Ala ProPro Val Pro Pro Thr Gly Asp Ser Gly Ala Pro Pro Val 580 585 590 595 CCGCCC ACG GGT GAC TCC GGG GCC CCC CCC GTG CCG CCC ACG GGT GAC 1983 Pro ProThr Gly Asp Ser Gly Ala Pro Pro Val Pro Pro Thr Gly Asp 600 605 610 TCCGGG GCC CCC CCC GTG CCG CCC ACG GGT GAC TCC GGG GCC CCC CCC 2031 Ser GlyAla Pro Pro Val Pro Pro Thr Gly Asp Ser Gly Ala Pro Pro 615 620 625 GTGCCG CCC ACG GGT GAC TCC GGC GCC CCC CCC GTG CCG CCC ACG GGT 2079 Val ProPro Thr Gly Asp Ser Gly Ala Pro Pro Val Pro Pro Thr Gly 630 635 640 GACGCC GGG CCC CCC CCC GTG CCG CCC ACG GGT GAC TCC GGC GCC CCC 2127 Asp AlaGly Pro Pro Pro Val Pro Pro Thr Gly Asp Ser Gly Ala Pro 645 650 655 CCCGTG CCG CCC ACG GGT GAC TCC GGG GCC CCC CCC GTG ACC CCC ACG 2175 Pro ValPro Pro Thr Gly Asp Ser Gly Ala Pro Pro Val Thr Pro Thr 660 665 670 675GGT GAC TCC GAG ACC GCC CCC GTG CCG CCC ACG GGT GAC TCC GGG GCC 2223 GlyAsp Ser Glu Thr Ala Pro Val Pro Pro Thr Gly Asp Ser Gly Ala 680 685 690CCC CCT GTG CCC CCC ACG GGT GAC TCT GAG GCT GCC CCT GTG CCC CCC 2271 ProPro Val Pro Pro Thr Gly Asp Ser Glu Ala Ala Pro Val Pro Pro 695 700 705ACA GAT GAC TCC AAG GAA GCT CAG ATG CCT GCA GTC ATT AGG TTT TAG 2319 ThrAsp Asp Ser Lys Glu Ala Gln Met Pro Ala Val Ile Arg Phe * 710 715 720CGTCCCATGA GCCTTGGTAT CAAGAGGCCA CAAGAGTGGG ACCCCAGGGG CTCCCCTCCC 2379ATCTTGAGCT CTTCCTGAAT AAAGCCTCAT ACCCCTAAAA AAAAAAAAA 2428 745 aminoacids amino acid linear protein 2 Met Leu Thr Met Gly Arg Leu Gln LeuVal Val Leu Gly Leu Thr Cys -23 -20 -15 -10 Cys Trp Ala Val Ala Ser AlaAla Lys Leu Gly Ala Val Tyr Thr Glu -5 1 5 Gly Gly Phe Val Glu Gly ValAsn Lys Lys Leu Gly Leu Leu Gly Asp 10 15 20 25 Ser Val Asp Ile Phe LysGly Ile Pro Phe Ala Ala Pro Thr Lys Ala 30 35 40 Leu Glu Asn Pro Gln ProHis Pro Gly Trp Gln Gly Thr Leu Lys Ala 45 50 55 Lys Asn Phe Lys Lys ArgCys Leu Gln Ala Thr Ile Thr Gln Asp Ser 60 65 70 Thr Tyr Gly Asp Glu AspCys Leu Tyr Leu Asn Ile Trp Val Pro Gln 75 80 85 Gly Arg Lys Gln Val SerArg Asp Leu Pro Val Met Ile Trp Ile Tyr 90 95 100 105 Gly Gly Ala PheLeu Met Gly Ser Gly His Gly Ala Asn Phe Leu Asn 110 115 120 Asn Tyr LeuTyr Asp Gly Glu Glu Ile Ala Thr Arg Gly Asn Val Ile 125 130 135 Val ValThr Phe Asn Tyr Arg Val Gly Pro Leu Gly Phe Leu Ser Thr 140 145 150 GlyAsp Ala Asn Leu Pro Gly Asn Tyr Gly Leu Arg Asp Gln His Met 155 160 165Ala Ile Ala Trp Val Lys Arg Asn Ile Ala Ala Phe Gly Gly Asp Pro 170 175180 185 Asn Asn Ile Thr Leu Phe Gly Glu Ser Ala Gly Gly Ala Ser Val Ser190 195 200 Leu Gln Thr Leu Ser Pro Tyr Asn Lys Gly Leu Ile Arg Arg AlaIle 205 210 215 Ser Gln Ser Gly Val Ala Leu Ser Pro Trp Val Ile Gln LysAsn Pro 220 225 230 Leu Phe Trp Ala Lys Lys Val Ala Glu Lys Val Gly CysPro Val Gly 235 240 245 Asp Ala Ala Arg Met Ala Gln Cys Leu Lys Val ThrAsp Pro Arg Ala 250 255 260 265 Leu Thr Leu Ala Tyr Lys Val Pro Leu AlaGly Leu Glu Tyr Pro Met 270 275 280 Leu His Tyr Val Gly Phe Val Pro ValIle Asp Gly Asp Phe Ile Pro 285 290 295 Ala Asp Pro Ile Asn Leu Tyr AlaAsn Ala Ala Asp Ile Asp Tyr Ile 300 305 310 Ala Gly Thr Asn Asn Met AspGly His Ile Phe Ala Ser Ile Asp Met 315 320 325 Pro Ala Ile Asn Lys GlyAsn Lys Lys Val Thr Glu Glu Asp Phe Tyr 330 335 340 345 Lys Leu Val SerGlu Phe Thr Ile Thr Lys Gly Leu Arg Gly Ala Lys 350 355 360 Thr Thr PheAsp Val Tyr Thr Glu Ser Trp Ala Gln Asp Pro Ser Gln 365 370 375 Glu AsnLys Lys Lys Thr Val Val Asp Phe Glu Thr Asp Val Leu Phe 380 385 390 LeuVal Pro Thr Glu Ile Ala Leu Ala Gln His Arg Ala Asn Ala Lys 395 400 405Ser Ala Lys Thr Tyr Ala Tyr Leu Phe Ser His Pro Ser Arg Met Pro 410 415420 425 Val Tyr Pro Lys Trp Val Gly Ala Asp His Ala Asp Asp Ile Gln Tyr430 435 440 Val Phe Gly Lys Pro Phe Ala Thr Pro Thr Gly Tyr Arg Pro GlnAsp 445 450 455 Arg Thr Val Ser Lys Ala Met Ile Ala Tyr Trp Thr Asn PheAla Lys 460 465 470 Thr Gly Asp Pro Asn Met Gly Asp Ser Ala Val Pro ThrHis Trp Glu 475 480 485 Pro Tyr Thr Thr Glu Asn Ser Gly Tyr Leu Glu IleThr Lys Lys Met 490 495 500 505 Gly Ser Ser Ser Met Lys Arg Ser Leu ArgThr Asn Phe Leu Arg Tyr 510 515 520 Trp Thr Leu Thr Tyr Leu Ala Leu ProThr Val Thr Asp Gln Glu Ala 525 530 535 Thr Pro Val Pro Pro Thr Gly AspSer Glu Ala Thr Pro Val Pro Pro 540 545 550 Thr Gly Asp Ser Glu Thr AlaPro Val Pro Pro Thr Gly Asp Ser Gly 555 560 565 Ala Pro Pro Val Pro ProThr Gly Asp Ser Gly Ala Pro Pro Val Pro 570 575 580 585 Pro Thr Gly AspSer Gly Ala Pro Pro Val Pro Pro Thr Gly Asp Ser 590 595 600 Gly Ala ProPro Val Pro Pro Thr Gly Asp Ser Gly Ala Pro Pro Val 605 610 615 Pro ProThr Gly Asp Ser Gly Ala Pro Pro Val Pro Pro Thr Gly Asp 620 625 630 SerGly Ala Pro Pro Val Pro Pro Thr Gly Asp Ala Gly Pro Pro Pro 635 640 645Val Pro Pro Thr Gly Asp Ser Gly Ala Pro Pro Val Pro Pro Thr Gly 650 655660 665 Asp Ser Gly Ala Pro Pro Val Thr Pro Thr Gly Asp Ser Glu Thr Ala670 675 680 Pro Val Pro Pro Thr Gly Asp Ser Gly Ala Pro Pro Val Pro ProThr 685 690 695 Gly Asp Ser Glu Ala Ala Pro Val Pro Pro Thr Asp Asp SerLys Glu 700 705 710 Ala Gln Met Pro Ala Val Ile Arg Phe 715 720 722amino acids amino acid linear protein NO Homo sapiens Mammary gland 3Ala Lys Leu Gly Ala Val Tyr Thr Glu Gly Gly Phe Val Glu Gly Val 1 5 1015 Asn Lys Lys Leu Gly Leu Leu Gly Asp Ser Val Asp Ile Phe Lys Gly 20 2530 Ile Pro Phe Ala Ala Pro Thr Lys Ala Leu Glu Asn Pro Gln Pro His 35 4045 Pro Gly Trp Gln Gly Thr Leu Lys Ala Lys Asn Phe Lys Lys Arg Cys 50 5560 Leu Gln Ala Thr Ile Thr Gln Asp Ser Thr Tyr Gly Asp Glu Asp Cys 65 7075 80 Leu Tyr Leu Asn Ile Trp Val Pro Gln Gly Arg Lys Gln Val Ser Arg 8590 95 Asp Leu Pro Val Met Ile Trp Ile Tyr Gly Gly Ala Phe Leu Met Gly100 105 110 Ser Gly His Gly Ala Asn Phe Leu Asn Asn Tyr Leu Tyr Asp GlyGlu 115 120 125 Glu Ile Ala Thr Arg Gly Asn Val Ile Val Val Thr Phe AsnTyr Arg 130 135 140 Val Gly Pro Leu Gly Phe Leu Ser Thr Gly Asp Ala AsnLeu Pro Gly 145 150 155 160 Asn Tyr Gly Leu Arg Asp Gln His Met Ala IleAla Trp Val Lys Arg 165 170 175 Asn Ile Ala Ala Phe Gly Gly Asp Pro AsnAsn Ile Thr Leu Phe Gly 180 185 190 Glu Ser Ala Gly Gly Ala Ser Val SerLeu Gln Thr Leu Ser Pro Tyr 195 200 205 Asn Lys Gly Leu Ile Arg Arg AlaIle Ser Gln Ser Gly Val Ala Leu 210 215 220 Ser Pro Trp Val Ile Gln LysAsn Pro Leu Phe Trp Ala Lys Lys Val 225 230 235 240 Ala Glu Lys Val GlyCys Pro Val Gly Asp Ala Ala Arg Met Ala Gln 245 250 255 Cys Leu Lys ValThr Asp Pro Arg Ala Leu Thr Leu Ala Tyr Lys Val 260 265 270 Pro Leu AlaGly Leu Glu Tyr Pro Met Leu His Tyr Val Gly Phe Val 275 280 285 Pro ValIle Asp Gly Asp Phe Ile Pro Ala Asp Pro Ile Asn Leu Tyr 290 295 300 AlaAsn Ala Ala Asp Ile Asp Tyr Ile Ala Gly Thr Asn Asn Met Asp 305 310 315320 Gly His Ile Phe Ala Ser Ile Asp Met Pro Ala Ile Asn Lys Gly Asn 325330 335 Lys Lys Val Thr Glu Glu Asp Phe Tyr Lys Leu Val Ser Glu Phe Thr340 345 350 Ile Thr Lys Gly Leu Arg Gly Ala Lys Thr Thr Phe Asp Val TyrThr 355 360 365 Glu Ser Trp Ala Gln Asp Pro Ser Gln Glu Asn Lys Lys LysThr Val 370 375 380 Val Asp Phe Glu Thr Asp Val Leu Phe Leu Val Pro ThrGlu Ile Ala 385 390 395 400 Leu Ala Gln His Arg Ala Asn Ala Lys Ser AlaLys Thr Tyr Ala Tyr 405 410 415 Leu Phe Ser His Pro Ser Arg Met Pro ValTyr Pro Lys Trp Val Gly 420 425 430 Ala Asp His Ala Asp Asp Ile Gln TyrVal Phe Gly Lys Pro Phe Ala 435 440 445 Thr Pro Thr Gly Tyr Arg Pro GlnAsp Arg Thr Val Ser Lys Ala Met 450 455 460 Ile Ala Tyr Trp Thr Asn PheAla Lys Thr Gly Asp Pro Asn Met Gly 465 470 475 480 Asp Ser Ala Val ProThr His Trp Glu Pro Tyr Thr Thr Glu Asn Ser 485 490 495 Gly Tyr Leu GluIle Thr Lys Lys Met Gly Ser Ser Ser Met Lys Arg 500 505 510 Ser Leu ArgThr Asn Phe Leu Arg Tyr Trp Thr Leu Thr Tyr Leu Ala 515 520 525 Leu ProThr Val Thr Asp Gln Glu Ala Thr Pro Val Pro Pro Thr Gly 530 535 540 AspSer Glu Ala Thr Pro Val Pro Pro Thr Gly Asp Ser Glu Thr Ala 545 550 555560 Pro Val Pro Pro Thr Gly Asp Ser Gly Ala Pro Pro Val Pro Pro Thr 565570 575 Gly Asp Ser Gly Ala Pro Pro Val Pro Pro Thr Gly Asp Ser Gly Ala580 585 590 Pro Pro Val Pro Pro Thr Gly Asp Ser Gly Ala Pro Pro Val ProPro 595 600 605 Thr Gly Asp Ser Gly Ala Pro Pro Val Pro Pro Thr Gly AspSer Gly 610 615 620 Ala Pro Pro Val Pro Pro Thr Gly Asp Ser Gly Ala ProPro Val Pro 625 630 635 640 Pro Thr Gly Asp Ala Gly Pro Pro Pro Val ProPro Thr Gly Asp Ser 645 650 655 Gly Ala Pro Pro Val Pro Pro Thr Gly AspSer Gly Ala Pro Pro Val 660 665 670 Thr Pro Thr Gly Asp Ser Glu Thr AlaPro Val Pro Pro Thr Gly Asp 675 680 685 Ser Gly Ala Pro Pro Val Pro ProThr Gly Asp Ser Glu Ala Ala Pro 690 695 700 Val Pro Pro Thr Asp Asp SerLys Glu Ala Gln Met Pro Ala Val Ile 705 710 715 720 Arg Phe 568 aminoacids amino acid linear protein NO Homo sapiens Mammary gland Peptide1..568 /label= Variant_C Lennart Blackberg, Lars Edlund, MichaelLundberg, Lennart Stromqvist, Mats Hernell, Olle Hansson RecombinantHuman Milk Bile Salt-stimulated Lipase J. Biol. Chem. 268 35 26692-26698Dec. 15-1993 4 Ala Lys Leu Gly Ala Val Tyr Thr Glu Gly Gly Phe Val GluGly Val 1 5 10 15 Asn Lys Lys Leu Gly Leu Leu Gly Asp Ser Val Asp IlePhe Lys Gly 20 25 30 Ile Pro Phe Ala Ala Pro Thr Lys Ala Leu Glu Asn ProGln Pro His 35 40 45 Pro Gly Trp Gln Gly Thr Leu Lys Ala Lys Asn Phe LysLys Arg Cys 50 55 60 Leu Gln Ala Thr Ile Thr Gln Asp Ser Thr Tyr Gly AspGlu Asp Cys 65 70 75 80 Leu Tyr Leu Asn Ile Trp Val Pro Gln Gly Arg LysGln Val Ser Arg 85 90 95 Asp Leu Pro Val Met Ile Trp Ile Tyr Gly Gly AlaPhe Leu Met Gly 100 105 110 Ser Gly His Gly Ala Asn Phe Leu Asn Asn TyrLeu Tyr Asp Gly Glu 115 120 125 Glu Ile Ala Thr Arg Gly Asn Val Ile ValVal Thr Phe Asn Tyr Arg 130 135 140 Val Gly Pro Leu Gly Phe Leu Ser ThrGly Asp Ala Asn Leu Pro Gly 145 150 155 160 Asn Tyr Gly Leu Arg Asp GlnHis Met Ala Ile Ala Trp Val Lys Arg 165 170 175 Asn Ile Ala Ala Phe GlyGly Asp Pro Asn Asn Ile Thr Leu Phe Gly 180 185 190 Glu Ser Ala Gly GlyAla Ser Val Ser Leu Gln Thr Leu Ser Pro Tyr 195 200 205 Asn Lys Gly LeuIle Arg Arg Ala Ile Ser Gln Ser Gly Val Ala Leu 210 215 220 Ser Pro TrpVal Ile Gln Lys Asn Pro Leu Phe Trp Ala Lys Lys Val 225 230 235 240 AlaGlu Lys Val Gly Cys Pro Val Gly Asp Ala Ala Arg Met Ala Gln 245 250 255Cys Leu Lys Val Thr Asp Pro Arg Ala Leu Thr Leu Ala Tyr Lys Val 260 265270 Pro Leu Ala Gly Leu Glu Tyr Pro Met Leu His Tyr Val Gly Phe Val 275280 285 Pro Val Ile Asp Gly Asp Phe Ile Pro Ala Asp Pro Ile Asn Leu Tyr290 295 300 Ala Asn Ala Ala Asp Ile Asp Tyr Ile Ala Gly Thr Asn Asn MetAsp 305 310 315 320 Gly His Ile Phe Ala Ser Ile Asp Met Pro Ala Ile AsnLys Gly Asn 325 330 335 Lys Lys Val Thr Glu Glu Asp Phe Tyr Lys Leu ValSer Glu Phe Thr 340 345 350 Ile Thr Lys Gly Leu Arg Gly Ala Lys Thr ThrPhe Asp Val Tyr Thr 355 360 365 Glu Ser Trp Ala Gln Asp Pro Ser Gln GluAsn Lys Lys Lys Thr Val 370 375 380 Val Asp Phe Glu Thr Asp Val Leu PheLeu Val Pro Thr Glu Ile Ala 385 390 395 400 Leu Ala Gln His Arg Ala AsnAla Lys Ser Ala Lys Thr Tyr Ala Tyr 405 410 415 Leu Phe Ser His Pro SerArg Met Pro Val Tyr Pro Lys Trp Val Gly 420 425 430 Ala Asp His Ala AspAsp Ile Gln Tyr Val Phe Gly Lys Pro Phe Ala 435 440 445 Thr Pro Thr GlyTyr Arg Pro Gln Asp Arg Thr Val Ser Lys Ala Met 450 455 460 Ile Ala TyrTrp Thr Asn Phe Ala Lys Thr Gly Asp Pro Asn Met Gly 465 470 475 480 AspSer Ala Val Pro Thr His Trp Glu Pro Tyr Thr Thr Glu Asn Ser 485 490 495Gly Tyr Leu Glu Ile Thr Lys Lys Met Gly Ser Ser Ser Met Lys Arg 500 505510 Ser Leu Arg Thr Asn Phe Leu Arg Tyr Trp Thr Leu Thr Tyr Leu Ala 515520 525 Leu Pro Thr Val Thr Asp Gln Gly Ala Pro Pro Val Pro Pro Thr Gly530 535 540 Asp Ser Gly Ala Pro Pro Val Pro Pro Thr Gly Asp Ser Lys GluAla 545 550 555 560 Gln Met Pro Ala Val Ile Arg Phe 565

1. A DNA molecule comprising: (a) a region coding for a polypeptidewhich is human BSSL or a biologically active variant thereof; (b) joinedto the 5′-end of said polypeptide coding region, a region coding for asignal peptide capable of directing secretion of said polypeptide fromPichia pastoris cells transformed with said DNA molecule; and (c)operably-linked to said coding regions defined in (a) and (b), themethanol oxidase promoter of Pichia pastoris or a functionallyequivalent promoter.
 2. A DNA molecule according to claim 1 wherein thesaid signal peptide is identical to, or substantially similar to, thepeptide with the amino acid sequence shown as amino acids −20 to −1 ofSEQ ID NO: 2 in the Sequence Listing.
 3. A DNA molecule according toclaim 1 wherein the said signal peptide comp rises a Saccharomycescerevisiae invertase signal peptide.
 4. A DNA molecule according to anyone of claims 1 to 3 encoding a biologically active variant of humanBSSL in which at least one of the repeat units of 11 amino acids, saidrepeated units being indicated in SEQ ID NO: 1, is deleted.
 5. A DNAmolecule according to any one of claims 1 to 4 coding for a polypeptidewhich has BSSL activity and an amino acid sequence which is at least 95%homologous with the sequence according to SEQ ID NO: 3 or SEQ ID NO: 4.6. A DNA molecule according to any one of claims 1 to 5 coding for apolypeptide which has the amino acid sequence according to SEQ ID NO: 3or SEQ ID NO:
 4. 7. A vector comprising a DNA molecule according to anyone of claims 1 to
 6. 8. A replicable expression vector according toclaim 7 which is capable of mediating expression of human BSSL, or abiologically active variant thereof, in Pichia pastoris cells.
 9. Avector according to claim 8 which is the plasmid vector pARC 5771 (NCIMB40721), pARC 5799 (NCIMB 40723) or pARC 5797 (NCIMB 40722).
 10. Hostcells of the genus Pichia transformed with a vector according to any oneof claims 7 to
 9. 11. Host cells according to claim 10 which are Pichiapastoris cells.
 12. Host cells according to claim 11 which are Pichiapastoris cells of the strain GS115.
 13. Host cells according to claim 12which are PPF-1[pARC 5771] (NCIMB 40721), GS115[pARC 5799] (NCIMB 40723)or GS115[pARC 5797] (NCIMB 40722).
 14. A process for the production of apolypeptide which is human BSSL, or a biologically active variantthereof, which comprises culturing host cells according to any one ofclaims 10 to 13 under conditions whereby said polypeptide is secretedinto the culture medium, and recovering said polypeptide from theculture medium.